Bioavailability of (-)-epicatechin upon intake of chocolate and cocoa in human volunteers

We evaluated the levels of (-)-epicatechin (EC) and its metabolites in plasma and urine after intake of chocolate or cocoa by male volunteers. EC metabolites were analyzed by HPLC and LC/MS after glucuronidase and/or sulfatase treatment. The maximum levels of total EC metabolites in plasma were reached 2 hours after either chocolate or cocoa intake. Sulfate, glucuronide, and sulfoglucuronide (mixture of sulfate and glucuronide) conjugates of nonmethylated EC were the main metabolites present in plasma rather than methylated forms. Urinary excretion of total EC metabolites within 24 hours after chocolate or cocoa intake was 29.8 ± 5.3% and 25.3 ± 8.1% of total EC intake. EC in chocolate and cocoa was partly absorbed and was found to be present as a component of various conjugates in plasma, and these were rapidly excreted in urine.     

Bioavailability of (-)-epicatechin upon intake of chocolate and cocoa in human volunteers

We evaluated the levels of (-)-epicatechin (EC) and its metabolites in plasma and urine after intake of chocolate or cocoa by male volunteers. EC metabolites were analyzed by HPLC and LC/MS after glucuronidase and/or sulfatase treatment. The maximum levels of total EC metabolites in plasma were reached 2 hours after either chocolate or cocoa intake. Sulfate, glucuronide, and sulfoglucuronide (mixture of sulfate and glucuronide) conjugates of nonmethylated EC were the main metabolites present in plasma rather than methylated forms. Urinary excretion of total EC metabolites within 24 hours after chocolate or cocoa intake was 29.8 ± 5.3% and 25.3 ± 8.1% of total EC intake. EC in chocolate and cocoa was partly absorbed and was found to be present as a component of various conjugates in plasma, and these were rapidly excreted in urine.     

Quantitative determination of unchanged cisplatin in rat kidney and liver by high-performance liquid chromatography

A quantitative analytical method for measuring unchanged cisplatin (CDDP) and high- and low-molecular-mass metabolites (fixed and mobile metabolites) in rat kidney and liver was developed. Unchanged CDDP, separated from fixed and mobile metabolites in tissue homogenates by consecutive procedures of fractionation and ultrafiltration, was determined by high-performance liquid chromatography (HPLC) with post-column derivatization. Although unchanged CDDP was found to be partly metabolized to fixed metabolites during the preparation of cytosolic ultrafiltrates, the recovery of unchanged CDDP gave a constant value (about 70%), which was independent of tissue type and CDDP concentration (from 1 to 10 μg/ml). The detection limit for unchanged CDDP in the cytosolic ultrafiltrate was 20 ng/ml, corresponding to a concentration detection limit of 65 ng Pt per g of tissue in the kidney and liver. The concentrations of fixed and mobile metabolites were determined as platinum concentrations in the tissue homogenate and in the cytosolic ultrafiltrate using atomic absorption spectrometry after correcting for transformation of unchanged CDDP to fixed metabolites. The distribution of unchanged CDDP, mobile metabolites and fixed metabolites in rat kidney and liver, after bolus injection of CDDP (5 mg/kg), was determined using this method.     

Characterisation of metabolites of the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline formed after incubation with isolated rat liver cells

The metabolism of 14C-labelled 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) was studied in suspensions of hepatocytes isolated from PCB-pretreated rats. The metabolites found after incubation of IQ/MeIQ (0.1 mM) with PCB-pretreated hepatocytes for 3 h were separated into three principal groups: ethyl acetate-extractable metabolites (2-4%), water soluble metabolites (94-98%) and covalently bound metabolites (0.4-0.5%). The water soluble metabolites were separated by HPLC. The metabolites were evaluated by beta-glucuronidase lability, sulphate incorporation and compared with glucuronides formed by microsomes. Mass spectroscopy and proton NMR were also run. The major metabolites formed were a N2-sulphamate, an O-sulphate in position 5 for IQ and 5 for MeIQ and an O-glucuronide in the same position. The MeIQ N2-sulphamate was much less abundant than the IQ N2-sulphamate. When compared with hepatocytes from uninduced rats, it was found that primarily the formation of ring-hydroxylated conjugates increased after PCB-pretreatment. The major ethyl acetate-extractable metabolites were the N2-acetyl derivatives and an unidentified metabolite. A small peak representing the 5-hydroxy-IQ or 5-hydroxy-MeIQ could also be seen in the HPLC chromatogram of the ethyl acetate extractable metabolites. All major water soluble products described in hepatocytes were also found in urine and bile of uninduced rats exposed to IQ/MeIQ in vivo.     

Metabolism of 5,6-epoxyeicosatrienoic acid by the human platelet. Formation of novel thromboxane analogs.

Radiolabeled cis-(+-)-5,6-epoxyeicosatrienoic acid (5(6)-EpETrE) was incubated with a suspension of isolated human platelets in order to study its metabolic fate. The epoxide slowly disappeared from the suspension and was completely metabolized within 30 min. After extraction and analysis by reverse-phase high performance liquid chromatography, seven metabolites were found. Addition of either indomethacin (0.01 mM, cyclooxygenase inhibitor) or BW755C (0.1 mM, cyclooxygenase/lipoxygenase inhibitor) to the incubations blocked the formation of four and six metabolites, respectively, 1,2-Epoxy-3,3,3-trichloropropane (inhibitor of microsomal epoxide hydrolase) failed to inhibit the formation of 5,6-dihydroxyeicosatrienoic acid (5,6-DiHETrE), a hydrolysis product of the precursor 5(6)-EpETrE. The metabolites were characterized by UV spectroscopy, negative ion chemical ionization liquid chromatography/mass spectrometry, gas chromatography/mass spectrometry and, in one instance, coelution with synthetic standard. Three primary platelet metabolites were structurally determined to be 5,6-epoxy-12-hydroxyeicosatrienoic acid, 5,6-epoxy-12-hydroxyheptadecadienoic acid, and a unique bicyclic metabolite, 5-hydroxy-6,9-epoxy-thromboxane B1, which originated from intramolecular hydrolysis of 5,6-epoxythromboxane-B1. This thromboxane analog was partially separated into stereoisomers and coeluted with the racemic synthetic standard in gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. Three other metabolites were characterized as 5,6,12-trihydroxyeicosatrienoic acid, 5,6,12-trihydroxyheptadecadienoic acid, and 5,6-dihydroxythromboxane-B1, and resulted from the hydrolysis of the corresponding epoxides rather than from the metabolism of 5,6-DiHETrE. The latter was not metabolized by platelet cyclooxygenase or lipoxygenase. The biosynthesis of two cyclooxygenase metabolites indicated the formation of unstable 5,6-epoxythromboxane-A1 as an intermediate precursor. Platelet aggregation was not induced by 5(6)-EpETrE, although responsiveness to arachidonic acid was reduced following preincubation with the epoxide. The platelet metabolites of 5(6)-EpETrE might be useful in assessing its in vivo production in humans.     

Quantitative determination of tulobuterol and its metabolites in human urine by mass fragmentography

A method is described for the simple and simultaneous determination of tulobuterol and its metabolites in human urine by gas chromatography-mass spectrometry. Quantification was achieved by single-ion monitoring at m/e 86 derived from trimethylsilyl-tulobuterol and its metabolites using a column packed with a mixed phase, 2% OV-1–2% QF-1 (1 : 1, w/w). The detection limits were estimated to be 2 ng/ml in urine for tulobuterol and 5 ng/ml for metabolites, respectively.     

Use of reverse-phase liquid chromatography, linked to tandem mass spectrometry, to profile the Calvin cycle and other metabolic intermediates in Arabidopsis rosettes at different carbon dioxide concentrations

A platform using reverse-phase liquid chromatography coupled to tandem mass spectrometry was developed to measure 28 metabolites from photosynthetic metabolism. It was validated by comparison with authentic standards, with a requirement for distinct and clearly separated peaks, high sensitivity and repeatability in Arabidopsis rosette extracts. The recovery of authentic standards added to the plant material before extraction was 80–120%, demonstrating the reliability of the extraction and analytic procedures. Some metabolites could not be reliably measured, and were extracted and determined by other methods. Measurements of 37 metabolites in Arabidopsis rosettes after 15 min of illumination at different CO₂ concentrations showed that most Calvin cycle intermediates remain unaltered, or decrease only slightly (<30%), at compensation point CO₂, whereas dedicated metabolites in end-product synthesis pathways decrease strongly. The inhibition of end-product synthesis allows high levels of metabolites to be retained in the Calvin cycle to support a rapid cycle with photorespiration.     

Identification of cannabichromene metabolites by mass spectrometry: identification of eight new dihydroxy metabolites in the rabbit

Metabolites of cannabichromene (CBC) produced by hepatic microsomal incubates from rabbits and mice were examined by gas chromatography/mass spectrometry (GC/MS) as trimethylsilyl (TMS) and (2H9)TMS derivatives. Most metabolites were hydroxylated compounds whose mass spectra gave very little information on metabolite structure as fragmentation was dominated by formation of the substituted chromenyl ion. This prevented charge localization and diagnostic fragmentation at the site of metabolic attack. This paper describes the identification of these metabolites by GC/MS techniques using both deuterium-exchange reactions and hydrogenation of the metabolites to tetrahydro derivatives; the latter method was used to suppress chromenyl ion formation and to enhance the relative abundance of diagnostic fragment ions. Twenty-one metabolites were identified. Metabolites were found hydroxylated in all positions of both aliphatic chains, with additional compounds formed by epoxidation and reduction of the aliphatic double bond in the methylpentenyl chain. Dihydroxy metabolites were hydoxylated in both the pentyl and methylpentenyl chains in positions common to those hydroxylated in the monohydroxy metabolites.     

少年足球运动员运动性疲劳后的差异代谢物筛选*

目的: 探索少年足球运动员在运动性疲劳后的差异代谢物变化。方法: 以12名男性少年足球运动员(14～16岁)为试验对象,用功率自行车建立包含有氧运动和无氧运动的训练模型:完成6 min 150 W负荷、60～65 r/min的踏骑运动和30 s的负荷按照测试者体重而设定的最大速度踏骑运动,中间休息1 min,重复运动3组,组间休息3 min;测定运动员每组运动后的最大摄氧量值和平均无氧功率值,采集运动训练前后的尿液样本,利用气相-质谱联用(GC-MS)法检测尿液样本,通过数据库筛选潜在的差异代谢物。结果: 与运动前相比较,少年足球运动员运动疲劳后的平均无氧功率显著下降(P＜0.05),筛选出25个差异代谢物,其中3个代谢物显著升高(P＜0.05,0.01),22个代谢物显著降低(P＜0.05,0.01);上述差异代谢物的相关代谢通路归属为甘氨酸-丝氨酸-苏氨酸代谢、三羧酸循环、酪氨酸代谢、氮代谢和甘油磷酯代谢通路。结论: 少年足球运动员发生运动性疲劳后,机体的代谢物:肌氨酸、L-别苏氨酸、肌酸、丝氨酸、琥珀酸、柠檬酸、4-羟基苯乙酸、羟胺和乙醇胺产生明显的变化,上述差异代谢物可作为少年足球运动员疲劳评价的指标。     

Analysis of naphthalene adduct binding sites in model proteins by tandem mass spectrometry

The electrophilic metabolites of the polyaromatic hydrocarbon naphthalene have been shown to bind covalently to proteins and covalent adduct formation correlates with the cytotoxic effects of the chemical in the respiratory system. Although 1,2-naphthalene epoxide, naphthalene diol epoxide, 1,2-naphthoquinone, and 1,4-napthoquinone have been identified as reactive metabolites of interest, the role of each metabolite in total covalent protein adduction and subsequent cytotoxicity remains to be established. To better understand the target residues associated with the reaction of these metabolites with proteins, mass spectrometry was used to identify adducted residues following (1) incubation of metabolites with actin and protein disulfide isomerase (PDI), and (2) activation of naphthalene in microsomal incubations containing supplemental actin or PDI. All four reactive metabolites bound to Cys, Lys or His residues in actin and PDI. Cys(17) of actin was the only residue adducted by all metabolites; there was substantial metabolite selectivity for the majority of adducted residues. Modifications of actin and PDI, following microsomal incubations containing (14)C-naphthalene, were detected readily by 2D gel electrophoresis and phosphor imaging. However, target modifications on tryptic peptides from these isolated proteins could not be readily detected by MALDI/TOF/TOF and only three modified peptides were detected using high resolution-selective ion monitoring (HR-SIM). All the reactive metabolites investigated have the potential to modify several residues in a single protein, but even in tissues with very high rates of naphthalene activation, the extent of modification was too low to allow unambiguous identification of a significant number of modified residues in the isolated proteins.     

Assessment of [18F]fluoroethylflumazenil metabolites using high-performance liquid chromatography and tandem mass spectrometry

A simple procedure using HPLC and tandem mass spectrometry has been developed for the determination of fluoroethylflumazenil metabolites. Samples were precipitated with acetonitrile, evaporated to dryness followed by reconstitution with methanol. As mobile phase, 50 mM ammonium formate–methanol (58:42, v/v) was used. The method is valid both for cold and radiolabelled metabolites. Various cold metabolites (hydroxylated and/or dealkylated) were identified in rat and human microsome preparations. Radiolabelled metabolites arise from two or more transformations including hydroxylation. The methodology developed can be applied for further characterisation of metabolites, and for the determination of non metabolised [¹⁸F]fluoroethylflumazenil in routine clinical analysis.     

液相串联质谱法同时定量检测生物样本中鞘脂类化合物

鞘脂类中的主要活性分子鞘氨醇1-磷酸（S1P）,可通过介导细胞增殖、分化和迁移等发挥广泛的生物学功能|同时,S1P可在相关酶的作用下转变为其它形式.本文旨在建立一种简便的鞘脂类的制备方法,并结合液相串联质谱（LC-MS/MS）快速检测生物样本中纳克水平的鞘脂类化合物. 采用甲醇沉淀或经典的脂质提取方法获得鞘脂类化合物,再采用LC-MS/MS在多反应监测模式（MRM）下进行定量分析. 实验结果表明,甲醇沉淀法可简便快捷地获得血浆或脂蛋白中鞘氨醇类化合物; S1P、二氢鞘氨醇（DH-S1P）和鞘氨醇（SPH）的定量限分别为110.5、215.6和44.3 pg;人血浆中S1P、DH-S1P和SPH的含量分别为257.8±49.4 nmol/L、93.5±17.3 nmol/L和44.6± 7.4 nmol/L, 鞘脂类化合物在人血浆脂蛋白上的分布存在显著性差异|在ApoE-/-小鼠血浆中S1P、DH-S1P和SPH的含量分别为590.1±78.2 nmol/L、197.8±60.6 nmol/L和35.4±16.7 nmol/L|每1×106个人脐静脉内皮细胞（EA.hy926）中,S1P和SPH的含量分别为103.7±21.8 pg和16.3±5.3 ng.甲醇沉淀法结合LC-MS/MS可简便快捷地对血浆和脂蛋白中的鞘脂类化合物进行定量检测. 该方法特别适用于大量生物样本中鞘脂类的快速定量分析.     

The methyl-5 alpha-dihydrotestosterones mesterolone and drostanolone; gas chromatographic/mass spectrometric characterization of the urinary metabolites.

Before including the detection of the methyl-5 alpha-dihydrotestosterones mesterolone (1 alpha-methyl-17 beta-hydroxy-5 alpha-androstan-3-one) and drostanolone (2 alpha-methyl-17 beta-hydroxy-5 alpha-androstan-3-one) in doping control procedures, their urinary metabolites were characterized by gas chromatography/mass spectrometry. Several metabolites were found after enzymatic hydrolysis and conversion of the respective metabolites to their trimethylsilyl-enol-trimethylsilyl ether derivatives. The major metabolites of mesterolone and drostanolone were identified as 1 alpha-methyl-androsterone and 2 alpha-methyl-androsterone, respectively. The parent compounds and the intermediate 3 alpha,17 beta-dihydroxysteroid metabolites were detected as well. The reduction into the corresponding 3 beta-hydroxysteroids was a minor metabolic pathway. All metabolites were found to be conjugated to glucuronic acid.     

Excretion of norsteroids’ phase II metabolites of different origin in human

The urinary phase II metabolites of norsteroids, 19-norandrosterone, 19-noretiocholanolone and 19-norepiandrosterone glucuronide and sulphate, were analyzed in samples collected during the pregnancy, following the administration of norsteroids or the consumption of edible parts of non-castrated pig and in athletes’ samples in which they were found during routine controls. The level of the sulfo- and glucuroconjugated metabolites was precisely determined by GC/HRMS, after selective hydrolysis. The goal was to evaluate whether the fine analysis of the norsteroid conjugates produced and excreted in different conditions would show a pattern that could be linked to their origin. The delta ¹³C values of the metabolites formed following the ingestion of edible parts of non-castrated pig were measured by isotope ratio mass spectrometry. Our results indicated that it is not possible to determine the origin of the urinary metabolites based upon the sole evaluation of the different metabolites and conjugates. The GC/C/IRMS is the only method permitting to distinguish between the exogenous and endogenous origin of the metabolites.     

Determination of two mebeverine metabolites, mebeverine alcohol and desmethylmebeverine alcohol, in human plasma by a dual stable isotope-based gas chromatographic-mass spectrometric method

A dual stable isotope-based GC-MS method was developed for the simultaneous determination of two metabolites of mebeverine, mebeverine alcohol and desmethylmebeverine alcohol, in human plasma. Plasma samples were treated with β-glucuronidase to cleave the glucuronide conjugates of both compounds prior to analysis. The treated plasma was prepared for analysis by solid-phase extraction using octadecylsilane cartridges. The isolated metabolites were derivatized and analyzed by GC-MS using selected-ion monitoring. Plots of peak-area ratio were linear with metabolite concentration from 2 to 200 ng/ml and the limit of detection for both metabolites was 0.5 ng/ml. The GC-MS methodology was applied to the analysis of plasma from human subjects following peroral administration of mebeverine. Pharmacokinetic parameters for both metabolites were determined and suggest that relative systemic mebeverine exposure may potentially be assessed using metabolite kinetics, if the latter subsequently are demonstrated to be linear with mebeverine dose.     

Simple and sensitive high-performance liquid chromatographic method for the determination of diltiazem and six of its metabolites in human plasma

A sensitive, specific and reproducible high-performance liquid chromatographic technique is described for the simultaneous determination in human plasma of diltiazem (DZ) and six of its primary and secondary metabolites which are products of N- and O-demethylation, deacetylation and N-oxidation. The method involves addition of excess KHCO₃ to 1 ml of plasma, followed by extraction with 4 ml of ethyl acetate. The organic layer was extracted with 0.01 M HCl and the aqueous layer was dried under nitrogen and then reconstituted with 0.002 M HCl. DZ and its metabolites were free from interference and were baseline-separated. Calibration curves were linear in the concentration range studied (5–500 ng/ml for all the species). The lower limit of quantification of the assay was 5 ng/ml for DZ and the metabolites. Inter-day and intra-day coefficients of variation were less than 10%. The applicability of this procedure is shown by evaluating the kinetics of DZ and its metabolites in three patients receiving chronic DZ therapy. N-Demethyldiltiazem, deacetyldiltiazem and N-demethyldeacetyldiltiazem were found to be the major metabolites, as previously described. Deacetyldiltiazem N-oxide was found in two of the patients. The other two known but unreported metabolites in human, O-demethyldeacetyldiltiazem and N,O-didemethyldeacetyldiltiazem, were found in the plasma of all three patients.     

Microbiological production of omeprazole metabolites by Cunninghamella elegans

Incubation of Cunninghamella elegans ATCC 9245 and the anti ulcer drug omeprazole allowed putative fungal metabolites to be isolated in sufficient quantities for structural elucidation. Three metabolites produced by the fungi were isolated using semi-preparative HPLC and their structures identified by a combination of LC/MS(n) and NMR experiments. These isolates will be used as reference standards in the confirmatory analysis of mammalian metabolites of this drug.     

Detoxification of naturally occurring chromenes in larvae of the generalist herbivore Spodoptera littoralis (Noctuidae)

Five naturally occurring chromenes from the Asteraceae including the insecticidal compounds precocene II (1) and encecalin (2) were administered to last instar larvae of Spodoptera littoralis via the food or by topical contact. Metabolites formed and excreted via the frass were analysed by GC-MS and by direct comparison with reference compounds obtained by partial synthesis. In total 28 different metabolites were identified, many of them reported here for the first time. All metabolites detected originated from phase I reactions (most probably catalysed by Cytochrom P-450-dependent monooxygenases) by hydroxylation, demethylation or reduction of the parent chromenes. The resulting metabolites can be regarded as detoxification products based on previous structure-activity studies. The increased polarity of the metabolites will furthermore facilitate their excretion by the larvae compared to the more apolar parent chromenes. The largest number of metabolites (eight for each compound) was detected following oral treatment with precocene II and encecalin respectively. 3-Monool as well as 3,4-trans-diol derivatives predominated in the frass of larvae treated with the latter compounds whereas the 6-hydroxyethyl derivatives were the major metabolites of the other chromenes investigated. The patterns of metabolites originating from precocene II or encecalin were the same following oral application or topical treatment.     

基于UPLC法测定离体大鼠及人源肠道菌对三七中人参皂苷代谢的差异

为探究人与大鼠肠道菌群对三七水煎液中三醇型人参皂苷Rg1、Re及二醇型人参皂苷Rb1、Rd体外代谢的差异性及发现其代谢产物原人参二醇PPD与原人参三醇PPT,实验利用UPLC方法测定三七水煎液分别与人、大鼠肠道菌群在厌氧条件下共培养24h后的孵育液中4种皂苷的含量及代谢产物PPD与PPT的含量。结果表明三七中含有三醇型人参皂苷Rg19.4500mg/g、Re1.8872mg/g,二醇型人参皂苷Rb18.5816mg/g、Rd1.9456mg/g。与人源肠道菌共培养后,三七中含有的二醇型、三醇型人参皂苷含量显著降低,重要的是,在培养液中检测到代谢产物PPD和PPT的存在,含量分别为0.2136mg/g及0.0344mg/g,与大鼠肠道菌共培养后,三七中含有的二醇型皂苷含量有轻微降低,而三醇型皂苷含量未见明显变化,但有少量PPT(0.0184mg/g)的生成。由此可见:在体外条件下,三七水煎液中人参皂苷会被人肠道菌群降解生成代谢产物PPD和PPT,而大鼠肠道菌群的降解产物却仅有PPT生成,二者存在种属差异。     

不饱和脂肪酸参与调控金针菇菌柄伸长的潜在分析

金针菇Flammulina filiformis菌柄是产量构成的重要组成部分,也是评价质量的重要指标。菌盖下方0.6-1.5cm是菌柄的伸长区,其他区段不伸长。为了研究菌柄伸长的调控机制,我们采用液相色谱-串联质谱联用（LC-MS/MS）技术对金针菇菌柄的伸长区、过渡区和非伸长区进行检测,结合层次聚类分析（HCA）、t检验等统计学方法对差异代谢物进行研究,并通过KEGG分析代谢物富集情况。结果显示,共有69种代谢物在伸长区、过渡区和非伸长区呈现梯度差异,其中52种代谢物含量为梯度性降低,17种代谢物为梯度性增加。这些差异代谢物主要归属于脂肪酸、氨基酸、核苷酸、生物碱等,其中脂肪酸及其衍生物占主导地位,尤其是油酸、花生四烯酸和肾上腺酸含量在菌柄伸长区显著高于其他区段。根据金针菇菌柄的代谢物积累特点,推测脂肪酸及其衍生物可能参与调控金针菇菌柄伸长。